The measurement of nuclear characteristics by automated image analysis is a powerful approach in the detection, diagnosis and prognosis of various disease states. These characteristics have traditionally included morphology, ploidy, texture and contextural features. One of the most successful embodiments of this technology has been applied to mass cervical cancer screening in the form of commercial imaging systems from Hologic™ (the ThinPrep® Imaging System) and Becton, Dickinson and Company (FocalPoint™). Other commercial systems include OralAdvance™ and LungSign™ from Perceptronix Medical Inc. Laboratories for the early detection of oral and lung cancer respectively.
Nuclear morphology generally pertains to shape and size measurements; such as those describing perimeter roundness. Ploidy is generally applied via a stoichiometric staining protocol (e.g. Thionin-Feulgen) to determine abnormal chromosome counts, termed aneuploidy, and involves measuring integrated optical density across the nuclear area. Texture analysis methods are generally considered as either statistical or structural. Both approaches produce descriptive measures of the spatial and intensity variation of a nucleus' internal structure, or chromatin pattern. Finally, contextual features measure the spatial distribution of inter-nuclear arrangements within a tissue structure.
All of these methods have been applied individually and in combination to successfully discriminate between normal and abnormal pathology in various tissue types. While the focus of this work has generally been applied to bright-field imaging, some research has also been conducted on the evaluation of such approaches to fluorescence microscopy.
However, traditional fluorophores suffer from several problems that reduce their utility in the application of such techniques to this modality. Photobleaching is a key issue that degrades the signal of the sample over time, even in the timeframe required for image capture. Sensitivity and specificity to target molecules (e.g., DNA) is also a limiting factor in traditional staining methodologies. For example, the widely used DAPI (4′,6-diamidino-2-phenylindole) nuclear counterstain is useful for locating the position and shape of cell nuclei, but does not bind specifically to render interpretable nuclear texture. Other fluorescent dyes used as DNA counterstains and markers include the Hoechst stains (e.g., Hoechst 33258 and Hoechst 33342) and propidium iodide. These materials, however, suffer from photo-induced degradation of photoluminescence intensity and spectral shift.
The existing prior art in the use of fluorescent dyes as DNA counterstains and markers utilizes small molecule organic and inorganic complexes. The application of a nanomaterial-based counterstain system provides a means to overcome the inherent flaws in the use of small molecule fluorophores due to its photostable optical characteristics. Quantum dot nanomaterials have been used mostly to detect DNA using FRET (Fluorescence Resonance Energy Transfer) or PET (Photoinduced Electron Transfer) based systems, (Dubeftret, Nature Materials (2005), 4(11): 797-798.) Another application employing nanomaterials is the use of quantum dots conjugated to nucleic acid-based probes that can hybridize to their complementary DNA sequence targets. (Bentolila et al., Cell Biochemistry and Biophysics (2006), 45(1):59-70.) The quantum dot acts as a visual reporter to target sequences. However, the application of such a stain is not compatible with the TMPRSS-ERG and HER2 FISH (fluorescence in situ hybridization) assays because a labeled DNA probe that can bind generally to DNA molecules may hybridize to the target gene, thereby preventing hybridization of the target gene probe and masking the presence of the target gene. The use of quantum dots with DNA interacting molecules to stain nuclear DNA in fixed cells and tissue has not been reported in the scientific literature.